Our goal is to understand the mechanisms underlying the recognition and repair of DMA damage. We use yeast as an experimental organism to continue our studies on three different aspects of this essential biological process. The specific aims of this proposal are focused on the following three subjects: (1) The Rad52 DNA repair protein: We will continue our molecular genetic characterization of the Rad52 DNA repair pathway using Rad52-fluorescent protein fusions. We will follow up on leads from our genomic screen of the yeast gene disruption library to study the role of novel helicases and other DNA metabolism genes in Rad52 focus formation. We will continue to explore Rad52 function with respect to post-translational modifications such as SUMO and phosphorylation, (2) Sml1, a negative regulator of RNR: We will continue to investigate the regulatory circuitry of the Rnr Inhibitor, SmI1. We will determine which pathways and what modifications regulate Smi1 degradation during S phase and after DNA damage. We have gained a wealth of information from our screen for genes that affect Sml1 protein levels after DNA damage. We will examine how spindle checkpoint proteins and kinetochore proteins are involved in this regulation. (3) The Top3/Sgs1 DNA topoisomerase/hellcase complex: We will continue to explore the genetic and biochemical interactions between Top3, Sgs1 and Rmi1. We will study a separation of function allele of Sgs1. We will further characterize the role of the Shu suppressor complex (Shu1/Shu2/Psy3/Csm2) in en-or-free repair. We will continue screening the yeast gene disruption library for additional genes that interact with Top3, Sgs1 and Rmi1 using a SPA - specific ploidy ablation, our newly developed method. It is these combined genetic and cell biological approaches to the many issues related to the recognition and repair of DNA damage In yeast that will continue to yield insights into this important biological process.